The role and impact of biotechnological production processes gained weight during the last years. Concurrently with the rising importance of biotechnological processes the complexity of the manufactured products steadily increases.
Possible host cells for the expression of heterologous proteins are HEK (Human Embryonal Kidney), HeLa (Henrietta Lacks), COS (SV40 transformed African Green Monkey kidney cells) and CHO (Chinese Hamster Ovary) cells, because heterologous proteins expressed in these hosts can be folded, processed, maturated by proteases, glycosylated, and sulfated according to the pattern in human cells (see e.g. Watson, E., et al., Glycobiol. 4 (1994) 227-37).
The Epstein-Barr-Virus (EBV) is a pathogen of human B lymphocytes. It belongs to the class of human herpes viruses (herpesviridae). B lymphocytes transformed by EBV may propagate unregulatedly (Miller, G., in Virology ed. by Fields, B., Raven Press, N.Y. (1985) 563-590). A characteristic feature of EBV-transformed cells is the expression of the so called Epstein-Barr-Virus-Nuclear-Antigen (EBNA) proteins. Of these, six different variants have been identified so far.
The EBNA-1 protein plays an important role in the replication cycle of the EBV nucleic acid in transformed cells. In combination with a second EBV element, the origin of replication (oriP), which acts in cis, the replication and maintenance of episomes within the cell is enabled (Lupton, S., and Levine, A. J., Mol. Cell. Biol. 5 (1985) 2533-2542; Yates, J. L., et al., Nature (London) 313 (1985) 812-815).
The oriP-segment is made up of two regions: the dyad symmetry element and the family of repeats. The first is a 65 bp sequence segment. It is separated in the virus' nucleic acid by circa 1000 bp from the family of repeats. This second segment is composed of a 30 bp sequence that is repeated 20 times (Hudson, G. S., et al., Virology 147 (1985) 81-98; Reisman, D., et al., Mol. Cell. Biol. 5 (1985) 1822-1832).
The 30-bp family of repeats possesses the ability to enhance transcription, whereas the dyad symmetry element plays a role in replication. The complete oriP-segment assists in maintaining the plasmid extrachromosomally.
The combination of the EBNA-1 protein, i.e. a trans-acting initiator protein, and oriP permits the construction of plasmids, which, once they have been introduced into a cell, are stably maintained as episomes and are stably replicated during cell proliferation (see e.g. Yates, J. L., et al., Nature (London) 313 (1985) 812-815; Reisman, D., et al., Mol. Cell. Biol. 5 (1985) 1822-1832). These two elements exploit the replication mechanism of the host cell for replication of the episome (Bode, J., et al., Gene Ther. Mol. Biol. 6 (2001) 33-46).
Krysan, P. J., et al. (Mol. Cell. Biol. 9 (1989) 1026-1033) demonstrated that plasmids comprising the family of repeats of the EBV origin of replication can be permanently retained in the cell nucleus of human cells. Therefore the presence of the EBNA-1 protein was sufficient; no other element of the EBV was required (see also Aiyar, A., et al., EMBO J. 17 (1998) 6394-6403; Hung, S. C., et al., PNAS 98 (2001) 1865-1870; Yates, J. L., in DNA Replication in Eukaryotic Cells, ed. by DePhamphilis, M. L., Cold Spring Harbor Laboratory, N.Y. (1996) pages 751-774; Yates, J. L., et al., J. Vir. 74 (2000) 4512-4522).
The EBNA-1 protein links the episome to the host cell's chromosome during episome replication and thereby assures the propagation of the episome during cell division.
Such a mutual relationship is also known for other viruses, e.g. the large T-antigen from the Simian Virus 40 (SV 40) and the E1/E2-proteins from the Bovine Papilloma Virus (BPV) (see e.g. Gilbert, D. M., et al., Cell 50 (1987) 59-68; DuBridge, R. B., et al., Mutagen. 3 (1988) 1-9; Lebkowski, J. S., et al., Mol. Cell. Biol. 4 (1984) 1951-1960).
The combination of the elements EBNA-1 and oriP of the EBV has been used for the preparation of not integrating, extrachromosomal, autonomously replicating episomes. Horlick et al., e.g., used HEK (Human Embryonic Kidney) cells stably expressing the EBNA-1 protein for the expression of CRHR (corticotrophin releasing hormone receptor) from a plasmid containing the EBV oriP (Horlick, R. A., et al., Prot. Exp. Purif. 9 (1997) 301-308).
In U.S. Pat. No. 4,686,186 a recombinant vectors and a eukaryotic host transformed thereby have been reported. The EBV elements oriP and EBNA-1 were combined on the recombinant vector.
In WO 2002/090533 a process for the production of recombinant proteins by transient transfection of suspension-grown human embryonic kidney cells (293 cell line and its genetic variants) is reported. Plasmids providing the EBV origin of replication are maintained extrachromosomally.
In WO 2004/053137 a method for the production of recombinant polypeptides and/or untranslated RNA molecules in host cells is reported. WO 2004/018506 reports compositions and methods applicable in a regulatable expression system that is transiently transfected into mammalian host cells.
US patent application 2002/0086419 reports a recombinant vector for stable persistence of exogenous DNA in eukaryotic host cells and the uses of the recombinant vector for long-term stable production of a gene product in the host cell.
An expression vector useful for transfection of a selected mammalian host cell is reported in U.S. Pat. No. 5,707,830. The expression vector contains an Epstein-Barr-Virus family of repeats, a copy of the EBNA-1 gene that can be functionally expressed in the host cell, a eukaryotic DNA fragment, which provides the ability of the vector to replicate in the host cell, and an expression cassette.
WO 2002/027005 reports an enhanced transfection system comprising an episomal maintenance system, a strong promoter/enhancer, a protein transactivation system and a DNA coding for a heterologous protein. The preferred cell lines should be non-rodent, because an oriP containing plasmid is not replicating efficiently and CHO cells, e.g., lack cellular factors for the transactivation system.
Wysokenski, D. A., and Yates, J. L., (J. Vir. 63 (1989) 2657-2666) mentioned that EBV plasmid replication does not cross species lines from cells of primates to cells of rodents. This is based on a missing interaction with a host protein involved in DNA replication, which is missing in rodents.
Tomiyasu, K-i., et al., Biochem. Biophys. Res. Commun. 253 (1998) 733-738, Mizuguchi, H., et al., FEBS Letters 472 (2000) 173-178, and WO 2005/024030 report plasmids containing both an oriP and EBNA-1 structural gene together with other elements.
U.S. Pat. No. 5,976,807 reports a method for producing recombinant eukaryotic cell lines expressing multiple proteins of interest. Transfected cells are obtained expressing an EBNA-1 protein and contain at least two transfected episomes.